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Ethical approval for the use of human tissue
Anonymized tumor tissues from angiosarcoma patients undergoing surgery were collected with informed consent. Ethical approval was obtained from the local certified Medical Ethics Committee of the Radboudumc, Nijmegen, The Netherlands (File number 2016-2686). All experiments were performed in accordance with relevant guidelines and regulations.
Ethical approval for the use of animals
All applicable (inter)national and institutional guidelines and regulations for the care and use of animals were followed. All procedures performed were in accordance with the ethical standards of the animal ethical committee of the Radboud University, Nijmegen, The Netherlands (Project# 2015-0109). The study is reported in accordance with the Animal Research Reporting of In Vivo (ARRIVE) guidelines.
In vivo growth
Female CB-17/lcr-Prkdcscid/Rj SCID or BALB/cAnNRj-Foxn1nu/Foxn1nu mice from Janvier labs (6–8 weeks old) were subcutaneously implanted with small tumor pieces (~ 3 mm in diameter) under anaesthesia via inhalation of isoflurane (2.5–3.0%). Tumor growth was monitored by biweekly caliper measurements in three dimensions (length (l), width (w) and height (h); all maximum diameter). Tumor size was calculated using the formula: 4/3π x l/2 × w/2 × h/2. Mice were euthanized by cervical dislocation at a maximal tumor volume of 2 cm3. Tumors were resected and used for passage into additional mice or for model characterization.
From previous experiments we knew that some xenografts take a long period of time to start growing. However, if after 6 months no tumor growth was detected the model was considered unsuitable.
Use of cryopreserved material
At each passage tumor pieces were stored in Medium 199 (ThermoFisher) with 10% DMSO at − 80 °C. We implanted these pieces again in two mice to check if we were able to regrow the tumor pieces in order to be able to; (1) stop passaging the tumors when no experiments were planned in order to spare mice, and (2) share the material with other researchers. To thaw tumor pieces, aliquots were warmed to 65 °C in a heated water bath and tumor tissue was washed in complete Medium 199 (without DMSO) and immediately implanted in mice.
Characterization of the model
Hematoxylin and Eosin (H&E) staining and immunohistochemical stainings for the angiosarcoma specific diagnostic markers ERG and CD31 were performed to characterize the model17. Immunohistochemical stainings were performed in the Lab Vision Autostainer 360 (ThermoFisher Scientific) by using the EnVision FLEX, pH High Link Kit (Dako) and monoclonal rabbit anti-ERG (1:500, clone EPR3864, Abcam) or monoclonal mouse anti-CD31 (1:100, clone JC70A, Dako).
TSO500 panel-based sequencing was performed as described previously to analyze genomic aberrations that were present in the model18,19.
In brief, genomic DNA was isolated from formalin-fixed paraffin-embedded (FFPE) tissue. Library preparation was performed using the hybrid capture-based TSO500 library preparation kit (Illumina) following the manufacturer’s protocol.
Libraries were sequenced on a NextSeq 500 (Illumina). Sequence data were processed and analyzed by the TruSight Oncology 500 Local App version (Illumina). Unique molecular identifiers (UMIs) were used in the analysis to determine the unique coverage at each position. Coverage tables and a variant call file for single- and multiple-nucleotide variants, including number and percentage of variant alleles, were provided. The R-package Xenofilter was used to exclude mouse sequences that disturbed the analysis of the patient-derived xenograft tissue20.
Genomic variants were filtered by excluding: (1) variants not overlapping with exons and splice site regions (− 8/ + 8) except those in the TERT promoter region, (2) synonymous variants, unless located in a splice site region, (3) variants present with a frequency > 0.1% in the control population represented in The Exome Aggregation Consortium (ExAC) version 0.2, and (4) variants with a variant allele frequency of < 5%.
Identified variants were interpreted using the software Alamut visual version 2.13 and in addition, the aggregated knowledge-based tools, ClinVar, OncoKB and InterVar were used to review specific variants. Variants were manually analyzed and classified based on the predicted pathogenicity into 5 classes: class 1, not pathogenic; class 2, unlikely pathogenic; class 3, variant of unknown significance; class 4, likely pathogenic; and class 5, pathogenic. The interpretation of pathogenicity for variants in tumor suppressor genes (TSGs) was based on three prediction tools (sorting intolerant from tolerant (SIFT), Polyphen-2 and Align-Grantham Variation Grantham Deviation (Align-GVGD).
Amplifications were called based on median coverage normalization as previously described18. A relative coverage ≥ 3 was considered gene amplification. The number of gene copies was estimated by using the relative coverage corrected for the percentage of tumor cells in the sample.
The corresponding original patient sample was subsequently analyzed by TSO500 panel-based sequencing to validate the results of the PDX model.